Cosmetic composition from fish hatching fluid, methods for its production and uses thereof for improving the cosmetic appearance of skin

ABSTRACT

The present invention relates to cosmetic compositions obtained or obtainable from fish hatching fluid, methods of producing said compositions and their use in various cosmetic applications to the skin, particularly for reducing or preventing the cosmetic appearance or prevalence of wrinkles, fine lines, hyperpigmentation, laxity, dry skin, scaling and/or transepidermal water loss in skin of a mammalian animal.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 371 of PCT/EP2013/077368 filed on Dec. 19, 2013,which claims the benefit of priority to Great Britain (GB) applicationNo. 1223330.0 filed on Dec. 21, 2012 under the provisions of 35 U.S.C.119 and the International Convention for the protection of IndustrialProperty, which are incorporated herein by reference.

The present invention relates to methods of preparing a compositioncomprising polypeptides or portions of polypeptides, which is derivablefrom fish hatching fluid, and its use in various applications to theskin. In particular, the composition is useful for altering, preferablyimproving, the cosmetic appearance of aged skin.

The skin is one of the more vulnerable organs of the body. Skin is inconstant interaction with external stimuli, directly or indirectly, andis frequently exposed to, and affected by, environmental agents. Infact, the skin can be seen as the first point of contact with theoutside world. This constant exposure can result in unpleasant and/orunwanted physical and visible changes to the skin, particularly to thecosmetic appearance of skin. Whilst such changes may not threaten thehealth of an individual, such changes may be physically uncomfortable orvisibly disagreeable. Indeed, because the skin is so visible, changes tothe appearance of skin can lead to psychological stress. There istherefore a continuing need and demand for effective treatments tomaintain, restore or improve the condition of the skin, and inparticular to restore the youthful appearance of skin.

Skin forms the largest organ of the body, accounting for about 12-16percent of a person's weight. It performs many vital roles as both abarrier and a regulating influence between the outside world and thecontrolled environment within our bodies.

Skin consists of 3 layers, namely the epidermis, dermis and subcutis.The epidermis is the uppermost, epithelial layer of the skin. It acts asa physical barrier, preventing loss of water from the body, andpreventing entry of substances and organisms into the body. Itsthickness varies according to body site.

The epidermis consists of stratified squamous epithelium, i.e. itconsists of layers of flattened cells. Skin, hair and nails arekeratinised, meaning they have a dead, hardened hydrophobic surface madeof a protein called keratin. Epidermis is made impermeable due to itscontents of extracellular lipids associated with keratinocytes,especially in the middle layer of the epidermis (stratum lucidum).Mucous membranes (e.g. of the oesophagus, oral pharyngeal cavity,reproductive organs, and others) are mainly non-keratinised and moist.The epidermis has three main types of cell, namely keratinocytes (skincells), melanocytes (pigment-producing cells) and Langerhans cells(immune cells). The Merkel cell is a fourth, less prevalent, epidermalcell.

The keratinocytes mature and differentiate with accumulation of keratinas they move outwards. They eventually fall or rub off. They form fouror five distinct strata, which from the most superficial to the deepestare (i) the Stratum corneum (horny layer) with dead, dried-out hardcells without nuclei, (ii) the Stratum granulosum (granular layer) withcells containing basophilic granules and outwardly separated fromstratum corneum by the thin stratum lucidum, (iii) the Stratumspinulosum (spinous, spiny or prickle cell layer) in which the cellsbecome increasingly flattened as they move upward and (iv) the Stratumbasale (basal layer) with columnar (tall) regenerative cells.

Immediately below the epidermis is the basement membrane, a specialisedstructure that lies between the epidermis and dermis.

The dermis is the fibrous connective tissue or supportive layer of theskin. The major fibres are collagen fibres and elastin which areinterwoven.

The subcutis is the fat layer immediately below the dermis andepidermis. It is also called subcutaneous tissue, hypodermis orpanniculus. The subcutis mainly consists of fat cells (adipocytes),nerves and blood vessels.

New epithelial skin cells are created in the skin's lower layer, thestratum granulosum. Over time, cells migrate to the surface of the skinand become more acidic. During their 30 day journey, they die and becomesaturated with keratin. Keratin and associated lipids are importantbecause they protect the skin from outside elements.

Many factors may contribute to the deterioration in the cosmeticappearance of skin including disease, injury, environmental factors,age, hormone levels, medication, externally applied or ingestedmaterials, genetic conditions or a combination of these and otherfactors. Age related deterioration in the cosmetic appearance of skin isa universal factor, particularly photoageing, i.e. Dermatoheliosis. Thisdeterioration can be seen in irregularities or abnormalities in theskin, which may appear as, e.g. dry skin, wrinkles, fine lines,increased laxity (sagging) or altered pigmentation.

Photoageing is a term used for the characteristic changes induced bychronic UVA and UVB exposure. The deterioration of biological functionsand ability to manage metabolic stress is one of the major consequencesof the ageing process. Ageing is a complex, progressive process whichalso leads to functional and aesthetic changes in the skin.

Photoageing is a process of ageing of the skin attributed to continuous,long-term exposure of skin to ultraviolet (UV) radiation ofapproximately 245-290 nm, which may be from natural or synthetic light.Photoageing is thus also known as ageing of the skin, particularly ofthe face, ears, neck and hands, caused by UVA and UVB rays.

Dry and/or scaling skin is one of the most common signs of ageing skin.Although certain individuals are more susceptible to dry and/or scalingskin, the appearance of dry and/or scaling skin can affect anyone,regardless of age, gender, or skin type.

Dry skin occurs when the skin's outer layer (the stratum corneum withthe stratum lucidum) is depleted of water, i.e. via trans-epidermalwater loss (TEWL). When this layer is well-moistened, it minimizes waterloss through the skin and helps keep out irritants, allergens, andgerms. However, when the stratum corneum dries out, its protectivefunction is reduced. This allows greater water loss, leaving skinvulnerable to environmental factors.

Ideally the stratum corneum has a water content of 10% to 30%. Thiswater imparts to the skin its soft, smooth, and flexible texture, i.e.the characteristics associated with the youthful appearance of skin. Thewater comes from the atmosphere, the underlying layers of skin, andsweat. Oil produced by skin glands and fatty substances produced by skincells act as natural moisturizers, allowing the stratum corneum to sealin water.

The body continuously loses water from the skin's surface by evaporation(TEWL). Under normal conditions, the rate of loss is slow, and the wateris adequately replaced. Characteristic signs and symptoms of dry skinoccur when the water loss exceeds the water replacement, and the stratumcorneum's water content falls below 10%.

Moisturizers which improve or eradicate dry and/or scaling skin, therebyimproving the cosmetic appearance of skin, are highly desirable. Whilstmany moisturizers are known in the art, there remains a need for naturalproducts which are effective yet gentle.

Epidermal cells exhibiting a undesired or excessive pigmentation, i.e.hyperpigmentation, e.g. liver spots, is another common sign of ageingskin. Traditionally exfoliation may be used to remove epidermal cellsthat are detrimental to the cosmetic appearance of skin.

Exfoliation removes the outer strata of epidermis to reveal the newerskin cells beneath. Exfoliation may be achieved by physical means (i.e.abrasion of the skin) or by chemical means. Chemical exfoliants includescrubs containing salicylic acid, glycolic acid, fruit enzymes, citricacid or malic acid and may be applied in high concentrations by adermatologist, or in lower concentrations in over-the-counter products.Chemical exfoliation may involve the use of products that contain alphahydroxy acids (AHAs) or beta hydroxy acids (BHAs), or enzymes that actto loosen the glue-like substances that hold the cells together at celljunctions, allowing them to ease away. This type of exfoliation isrecommended for people treating acne.

The greatest disadvantage to exfoliation is the high price of some ofthe products and methods used to achieve it. Exfoliation will lead tosome initial redness to the skin. Near the end of chemical peels, theskin will frost, with colours varying from a bright white to gray on theskin surface.

Hence, effective methods to reduce hyperpigmentation of skin, which aregentler on the skin than exfoliation, are therefore desirable.

There thus remains a need for products and treatments suitable forpromoting the aesthetic appearance of skin. In other words, productsfor, and methods of, improving the cosmetic appearance of skin aredesirable. In particular, there is a demand for products and methods forrestoring the youthful appearance to aged skin and/or combating thesigns of ageing skin.

A composition comprising molecules, namely polypeptides or portions ofpolypeptides, which are found in fish hatching fluid have surprisinglynow been found to be remarkably effective at improving the cosmeticappearance of skin, particularly reducing the physical signs or symptomsassociated with ageing skin.

Hatching of embryos from oviparous vertebrate organisms, e.g. fish,amphibians, birds and reptiles, is facilitated by various enzymes,typically known as hatching enzymes, which are capable of partially orfully degrading the proteinaceous parts of the eggshell. Oocytes of allvertebrates have characteristic extracellular envelopes, known asvitelline envelopes, eggshells or chorion (used interchangeably herein),which are made up by the cross-linkage of various polypeptides.Proteases with different specificities act on the chorion to soften,erode and/or breakdown (i.e. degrade) the eggshell and facilitate therelease of the embryo. Hence, fluid released from the egg during thehatching process and/or the fluid in which the embryo hatches (i.e.hatching fluid) comprises a multitude of polypeptides and portions ofpolypeptides, i.e. degradation products.

Compositions comprising proteins and portions of polypeptides, which arederived from Salmonidae hatching fluid have surprisingly been found tohave pronounced effects on the cosmetic appearance of skin. Whilst notwishing to be bound by theory, the Examples demonstrate thatcompositions comprising Salmonidae hatching fluid polypeptides andportions of polypeptides are capable of restoring the youthfulappearance of skin. Hatching fluid from other fish contains polypeptidesthat are functionally equivalent to the polypeptides found in hatchingfluid from Salmonidae. It is thought that the combination ofpolypeptides and portions of polypeptides in the compositions definedherein (which are thought to comprise at least 50 different polypeptidesor portions of polypeptides) may interact with different types ofproteins present in the dermis and epidermis of the skin. It is believedthat the combination of polypeptides and portions of polypeptides maywork in synergy and that these interactions may be, at least in part,responsible for the effects of the composition on the cosmetic/aestheticappearance of the skin.

Accordingly, at its broadest, the invention can be seen to provide acomposition comprising polypeptides and portions of polypeptidesderivable from fish hatching fluid, wherein the composition is notobtained or obtainable from Salmonidae hatching fluid. In particular thecomposition is for use in, or in methods for, promoting the aestheticappearance of skin. In other words, the composition as described hereinis for use in, or in methods for, improving the cosmetic appearance ofskin. In a particularly preferred aspect, the invention may be seen asproviding a composition comprising polypeptides and portions ofpolypeptides derivable from fish hatching fluid, except Salmonidaehatching fluid, as described herein for use in, or in methods for,restoring the youthful appearance to aged skin and/or combating thesigns of ageing skin. The composition referred to above is also referredto herein as a “hatching fluid extract”. In addition to polypeptides andportions of polypeptides, said extract may comprise nativenon-proteinaceous material.

It will be evident from the disclosures below that a compositioncomprising polypeptides and portions of polypeptides derivable from fishhatching fluid, which is not obtained or obtainable (i.e. derived) fromSalmonidae hatching fluid, as described herein may be provided as acosmetic composition, which comprises one or more pharmaceuticallyacceptable excipients and/or diluents.

Thus, in one aspect the present invention provides a method of preparinga cosmetic composition as described herein from fish hatching fluid,wherein said hatching fluid is not Salmonidae hatching fluid, comprisingat least the steps of:

a) suspending fish eggs, wherein said eggs are not Salmonidae eggs, in aminimal volume of water (e.g. equivalent to the volume of the eggs orless);

b) inducing synchronized, rapid hatching of said eggs (preferably suchthat hatching is complete within less than 6 hours for more than 80% ofthe embryos);

c) optionally filtering the hatched eggs to obtain hatching fluid; and

d) filtering the hatching fluid of b) or c) to obtain the composition,wherein the step of filtering the hatching fluid comprises at least thesteps of:

(i) filtering the hatching fluid using a filter with a pore size of atleast 5 μm, preferably 5-15 μm, and particularly preferably a pore sizeof 7 μm, and collecting the filtrate;

(ii) filtering the filtrate from step (i) using a filter with a poresize of 0.30-0.60 μm, preferably a pore size of 0.35-0.55 μm,particularly preferably 0.40-0.50 μm, most preferably 0.45 μm, andcollecting the filtrate;

(iii) exchanging the water in the filtrate from step (ii) with apharmaceutically or cosmetically acceptable buffer;

(iv) filtering the solution obtained from step (iii) using a filter witha pore size of 0.15-0.30 μm, preferably a pore size of 0.22 μm andcollecting the filtrate; and

(v) preparing said cosmetic composition from the filtrate from step(iv).

As referred to herein, “hatching fluid” is the fluid released from eggsduring the process of hatching and may be in a crude, diluted orfiltered form. The crude hatching fluid refers to undiluted, untreatedfluid. Diluted hatching fluid refers to hatching fluid which may havebeen mixed with other fluid during or after hatching.

The present invention also provides a cosmetic composition obtained orobtainable by the method described herein.

The step of exchanging the water in the filtrate may be performed usingany suitable method known in the art, e.g. diafiltration or dialysis. Ina particularly preferred embodiment, this step is performed usingdiafiltration using a filter with an exclusion size of less than 15 kDa,preferably 10 kDa or less, e.g. 9, 8, 7, 6, 5, 4, 3 kDa or less.

Diafiltration uses ultrafiltration membranes to remove e.g. salts orother unwanted or undesirable microsolutes from a solution or as a wayof exchanging the solvent, e.g. buffer, of a solution. Small moleculesare separated from a solution while retaining larger molecules in theretentate (the material which does not pass through the filter).Microsolutes and solvents, e.g. water, are generally easily washedthrough the membrane. Typically about 3 volumes of diafiltration solvent(e.g. phosphate buffered saline) will eliminate 95% of the microsolute.Thus, the above filtrate from step (ii) is initially processed bydiafiltration and this results in the concentration of the retentate asa proportion of the solution (which contains the solubleimpurities/unwanted fraction of the hatching fluid) passes through themembrane. The retentate is then diluted with a pharmaceuticallyacceptable buffer, e.g. 0.5 mM Sodium phosphate and 1 mM Sodiumchloride, phosphate buffered saline etc. The diluted retentate may besubjected to repeated rounds of diafiltration, if necessary. Typically,prior to step (iv) the retentate is diluted such that the filtrate fromstep (iv) has an enzymatic activity of 3000-5000 mU/L, preferably3000-4000 mU/L and most preferably about 3400 mU/L. The enzymaticactivity of the filtrate may be measured by the capacity of the filtrateto cleave the Factor Xa chromogenic substrate(CH₃OCO-D-CHA-Gly-Arg-pNA-AcOH, Sigma aldrich product number:F3301-25MG). Prior to the step of diafiltration the hatching fluid maycomprise an enzymatic activity in the range of 10 to 150,000 mU/L. Oneunit (1 U) may be defined as the amount of the enzyme required tocatalyze the conversion of 1 μmol of substrate per minute.

The Factor Xa chromogenic substrate (Sigma-Aldrich) is cleaved by anenzyme present in the hatching fluid yielding a yellow product that canbe measured conveniently using spectrophotometrical analysis at awavelength of 405 nm. A typical assay comprises the addition of 100 μlhatching fluid solution, obtainable from step (iii) of the above method,to 600 μl substrate solution, comprising 10 μl Factor Xa chromogenicsubstrate (10 mg/ml in milli-q or distilled water), 70 μl 0.2 M Tris-HClpH 8.5 and 520 μl dH₂O. Conveniently the change in absorbance may bemeasured for 5-20 minutes (or up to an hour for samples with lowenzymatic activity), typically 10 minutes. The result is multiplied withan appropriate factor, e.g. 10 (for a 10 minute assay) to get the enzymeactivity per 1 ml of sample. Other appropriate and equivalent substratesmay be used to determine the activity of the hatching fluid.

As mentioned above, in some embodiments it may be advantageous tosynchronize the step of egg hatching to maximize the amount of hatchingfluid obtained, particularly the amount of the desired polypeptides orportions thereof in the hatching fluid, for purification. Synchronizedhatching may be achieved by any suitable method known in the art. Forinstance, some eggs may be synchronized using photo-manipulation, e.g.transferring eggs from the light (which inhibits hatching) in toconditions with no light. Manipulation of the temperature of the eggs,e.g. the temperature of the solution in which the eggs hatch,deoxygenation of the hatching environment, e.g. deoxygenation of thesolution in which the eggs hatch (Oppen-Berntsen et al. 1990,Aquaculture, 86, pp. 417-430), increasing the amount of carbon dioxidein the hatching environment, and stimulation of the eggs usingelectricity can also be used to cause synchronized hatching. In someembodiments, synchronized hatching may be achieved using pheromones,e.g. peptide pheromones capable of affecting, i.e. stimulating, embryodevelopment and hatching. As noted above, the eggs may be suspended in aminimal volume of water, which may be equivalent to the volume of eggsor less, e.g. for every 1 ml of eggs a suspending liquid of ≤1, 0.75,0.5, 0.25 ml may be used, e.g. from 0.5 to 1 ml. In preferredembodiments synchronized, rapid hatching of said eggs, is such thathatching is complete within less than 6 hours for more than 80% of theembryos. In particularly preferred embodiments, hatching is completewithin less than 5, 4, 3 or 2 hours, such as 0.5-6 hours, 1-5 hours,1.5-4 hours, 2-3 hours, e.g. 1-2 hours. Furthermore, in some embodimentshatching is complete within the periods stated above for more than 85%,90%, or 95% of the embryos, e.g. more than 90, 91, 92, 93, 94, 95, 96,97, 98 or 99% of the embryos.

In some embodiments it may be advantageous to dilute the hatching fluidto facilitate the subsequent purification steps, e.g. to reduce theviscosity of the hatching fluid. Thus, the method may comprise a furtherstep of diluting the hatching fluid prior to step (d). Preferably thefiltrate may be diluted by a factor of at least 0.1, 0.2, 0.5, 0.75,1.0, 1.5, 2, 3, 4, 5, 10, 15, 20, 50, 100, 1000, 5000 or 10000.

The method of preparing a cosmetic composition described above resultsin an enriched preparation which is preferably substantially free of anycontaminating components derived from the source material or materialused in the isolation procedure, e.g. components other than thepolypeptides or portions of polypeptides comprised in the crude hatchingfluid. In a preferred embodiment the composition may be enriched to adegree of purity of more than 30, 40, 50 or 60%, e.g. >70, 80 or 90%,purity as assessed w/w (dry weight) of the polypeptides and portions ofpolypeptides in comparison to the starting hatching fluid, i.e. 90%purity refers to a loss of 90% of the starting material (contaminatingcomponents) through the course of the method of preparation. However,compositions may be used which have lower purity, e.g. retain more than40, 50, 60, 70, 80 or 90% of the starting material.

Whilst the filtrate may itself form the cosmetic composition, optionallythe product (the filtrate of step (iv)) obtained or obtainable from theabove method may be diluted (or concentrated) to an appropriateconcentration to produce the cosmetic composition and/or prior to itsuse in the methods and uses of the invention in step (v). Thus, themethod may comprise a further step of diluting (or concentrating) thecomposition. Preferably the filtrate may be diluted (or concentrated) bya factor of at least 1.5, 2, 3, 4, 5, 10, 15, 20, 50, 100, 1000, 5000 or10000. Particularly preferably the final composition comprises 0.5-10%,e.g. 0.5-5%, preferably 0.5-3% (e.g. 1 or 3%) of the filtrate of step(iv). In a particularly preferred embodiment, the solution from step(iii) of the above method is diluted or concentrated to achieve asolution with an enzymatic activity of 1000-10000 mU/L as measured bythe above described method. Preferably the solution, and therefore thefiltrate from step (iv) comprises an activity of 2000-10000, 3000-9000,3000-7000, 3000-6000, 3000-5000 or 3000-4000 mU/L. Most preferably thesolution comprises an activity of about 3400 mU/L.

Optionally, one or more pharmaceutically acceptable excipients and/ordiluents may be added to the product obtained or obtainable from theabove method. Thus, the method may comprise a further step of adding oneor more pharmaceutically acceptable excipients and/or diluents to thecomposition or combining the composition with one or morepharmaceutically acceptable excipients and/or diluents during step (v).Alternative or additional preparation method steps include changing ormodifying the solvent, e.g. pH, ion concentration etc.

Other pharmaceutically acceptable components or ingredients may be addedto the product obtained or obtainable from the above method, e.g. duringstep (v). The one or more other components may be active components,i.e. components that have an effect on the skin, preferably that alsoact to promote the aesthetic appearance of skin or improve the cosmeticappearance of skin, e.g. in the cosmetic indications described herein.Thus, alternatively or additionally, the method may comprise a furtherstep of adding one or more pharmaceutically acceptable active componentsto the composition or combining the composition with one or morepharmaceutically acceptable active components in step (v).Pharmaceutically acceptable active components may include minerals,vitamins, enzymes, proteins, peptides, amino acids, lipids,antioxidants, polysaccharides, substances suitable as sunscreen filters,chemical exfoliants, extracts and mixtures thereof, as described in moredetail below.

The cosmetic composition obtained or obtainable from the above methodsis suitable for use in the methods of the invention, as describedelsewhere herein. In particular, the cosmetic composition is for use inimproving the cosmetic appearance of skin in a mammalian animal.

The invention also provides a method for improving the cosmeticappearance of skin of a mammalian animal wherein a cosmetic compositionas defined above is administered to said animal.

A further aspect of the invention is the use of a cosmetic compositionas defined above in the manufacture of a medicament for improving thecosmetic appearance of skin of a mammalian animal.

“Polypeptides” as referred to herein are molecules with preferably morethan 50, 100, 150, 200 or 250 residues and/or less than 500, 400, 300,200 or 100 residues or a range selected therefrom. As referred to hereina “portion” preferably comprises at least 30, 40, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,240 or more amino acids of the sequence from which it is derived. Saidportion may be obtained from a central or N-terminal or C-terminalportions of the sequence.

The compositions as defined herein may be obtained from any fish eggsexcept Salmonidae eggs, i.e. the compositions of the present inventionare not obtained or obtainable from Salmonidae eggs, e.g. eggs from thesub-families Salmo and Oncorhynchus, such as Atlantic salmon (Salmosalar) or Pacific salmon (Oncorhynchus masou). In general, fish lay eggswhich undergo little or no other embryonic development within the motherand fish eggs therefore represent a useful source of hatching fluid fromwhich the cosmetic composition of the invention may be obtained orobtainable by the method described herein. Accordingly, suitable fishthat may provide eggs as the starting material for the methods of theinvention include any fish of the Teleostei infraclass, which is one ofthree infraclasses of the class Actinopterygii, except fish from theSalmondiae family. Hence, the fish may be selected from a fish of anySuperorder selected from the list consisting of Osteoglossomorpha,Elopomorpha, Clupeomorpha, Ostariophysi, Protacanthopterygii (excludingfish from the Salmonidae family), Stenoptetygii, Cyclosquamata,Scopelomorpha, Lampridiomorpha, Polymyxiomorpha, Paracanthopterygii andAcanthopterygii.

In some embodiments, the fish may be selected from a fish of any Orderselected from the list consisting of Osteoglossiformes, Hiodontiformes,Elopiformes, Albuliformes, Notacanthiformes, Anguilliformes,Saccopharyngiformes, Clupeiformes, Gonorynchiformes, Cypriniformes,Characiformes, Gymnotiformes, Siluriformes, Argentiniformes,Salmoniformes (excluding fish from the Salmonidae family), Esociformes,Osmeriformes, Ateleopodiformes, Stomiiformes, Aulopiformes,Myctophiformes, Lampriformes, Polymixiiformes, Percopsiformes,Batrachoidiformes, Lophiiformes, Gadiformes, Ophidiiformes,Mugiliformes, Atheriniformes, Beloniformes, Cetomimiformes,Cyprinodontiformes, Stephanoberyciformes, Beryciformes, Zeiformes,Gobiesociformes, Gasterosteiformes, Syngnathiformes, Synbranchiformes,Tetraodontiformes, Pleuronectiformes, Scorpaeniformes Perciformes andAcipenseriformes.

In preferred embodiments the fish may be selected from a fish of anyOrder selected from the list consisting of Salmoniformes (excluding fishfrom the Salmonidae family), Cypriniformes, Perciformes, Siluriformes,Mugiliformes and Acipenseriformes.

In particularly preferred embodiments the fish may be selected from afish of any Family selected from the list consisting of Cyprinidae,Cichlidae, Pangasiidae, Sciaenidae, Serranidae, Carangidae, Sparidae,Lateolabracidae, Moronidae, Mugilidae, Oryziinae, Latidae, Eleotridaeand Acipenseridae.

Thus, in some embodiments, the fish may be a bony-tongued fish, amooneye, a goldeye, a ladyfish, a tarpon, a bonefish, a halosaur, aspiny eel, a true eel, a gulper, a gulper eel, a herring, an anchovy, amilkfish, a barb, a carp, a danio, a goldfish, a loach, a minnow, arasbora, a characin, a pencilfish, a hatchetfish, a piranha, a tetra, anelectric eel, a knifefish, a catfish, a barreleye, a slickhead, a pike,a smelt, a galaxiid, a jellynose fish, a bristlemouth, a marinehatchetfish, a Bombay duck fish, a lancetfish, a lanternfish, anoarfish, an opah, a ribbonfish, a beardfish, a cavefish, a trout-perch,a toadfish, an anglerfish, a cod, a pearlfish, a Silver arowana, amullet, a silverside, a rainbowfish, a flyingfish, a whalefish, alivebearer, a killifish, a ridgehead, a fangtooth, a pineconefish, adory, a clingfish, a stickleback, a pipefish, a seahorse, a swamp eel, afilefish, a pufferfish, a flatfish, a scorpionfish, a sculpin, ananabantid, a bass, a cichlid, a goby, a gourami, a mackerel, a perch, ascat, a whiting or a wrass.

In particularly preferred embodiments the fish may be any speciesselected from Grass carp (Ctenopharyngodon idella), Silver carp(Hypophthalmichthys molitrix), Catla (Catla catla), Common carp(Cyprinus carpio), Bighead carp (Hypophthalmichthys nobilis), Cruciancarp (Carassius carassius), Nile tilapia (Oreochromis niloticusniloticus), Pangas catfish (Pangasius pangasius), Roho labeo (Labeorohita), Large yellow croaker (Larimichthys crocea), Greasy grouper(Epinephelus tauvina), Japanese amberjack (Seriola quinqueradiata),Gilthead seabream (Sparus aurata), Japanese seabass (Lateolabraxjaponicus), European seabass (Dicentrarchus labrax), Silver seabream(Chrysophrys auratus), Flathead grey mullet (Mugil cephalus), Barramundi(Lates calcarifer), Marble goby (Oxyeleotris marmorata), Mozambiquetilapia (Oreochromis mossambicus), Japanese rice fish (Orzyias latipes),Zebrafish (Danio rerio), Siberian sturgeon (Acipenser baerii) and Danubesturgeon (Acipenser gueldenstaedtii).

In some embodiments, the cosmetic compositions may be obtained fromhatching fluid from more than one type of egg, i.e. eggs from more thanone type of fish. For instance, the hatching fluid from two or moretypes of egg could be used in the method described herein to obtain thecosmetic composition of the invention. Hence, the method of theinvention may include a step of combining the hatching fluid collectedfrom the hatched eggs of one or more fish, e.g. before or afterfiltration. Hence, the eggs from the fish described herein may be viewedas natural biological variations of the starting material.

The cosmetic compositions described herein are for use in vivo asdiscussed herein.

By “pharmaceutically acceptable” or “physiologically acceptable” or“cosmetically acceptable” is meant that the ingredient must be suitablefor cosmetic application and compositions. The ingredients also must becompatible with other ingredients in the composition as well asphysiologically acceptable to the recipient.

The active ingredient, i.e. the composition obtainable by the methoddescribed above, for administration may be appropriately modified foruse in a cosmetic composition. For example the composition used inaccordance with the invention may be stabilized against degradation forexample by the use of appropriate additives such as salts ornon-electrolytes, acetate, SDS, EDTA, citrate or acetate buffers,mannitol, glycine, HSA or polysorbate.

The compositions obtained by the methods described herein may be presentin the compositions for the cosmetic uses as the sole active ingredientor may be combined with other ingredients, particularly other activeingredients, e.g. to augment the cosmetic effect (as described above) orto make the composition more appealing to the consumer. In someembodiments, compositions obtained by the methods of the invention thatare derived from different sources of hatching fluid may be combined.

As mentioned above, the compositions described herein exhibit propertiesthat are useful in improving the cosmetic appearance of skin,particularly of aged skin, e.g. photo-aged skin.

The composition described herein may also comprise impurities, e.g.after the preparation of said composition from one of the abovedescribed natural sources. In compositions as described herein, thevarious polypeptides or portions of polypeptides derivable from fishhatching fluid may be present (in combination) in the range 0.0001 to50% w/w of the cosmetic composition prepared according to the abovedescribed method. Preferably said polypeptides or portions ofpolypeptides derivable from fish hatching fluid are present (incombination) at a range of 0.0001 to 10% w/w of the cosmetic composition(or up to 10-40%), e.g. 0.0001 to 5%, 0.0001 to 3%, 0.0001 to 2%, 0.0001to 1%, 0.0001 to 0.5%, 0.0001 to 0.1% w/w of the cosmetic compositionprepared according to the above method (e.g. at 0.01 to 0.1% w/w or0.0001 to 0.001 w/w if diluted in the final step). Accordingly,individual polypeptides or portions of polypeptides derivable from fishhatching fluid may be present at the range of 1×10⁻⁶ to 10% w/w of thecosmetic composition. In some embodiments said individual polypeptidesor portions of polypeptides derivable from fish hatching fluid may bepresent at the range 1×10⁻⁶ to 5% w/w of the cosmetic composition, e.g.1×10⁻⁶ to 4%, 1×10⁻⁶ to 3%, 1×10⁻⁶ to 2%, 1×10⁻⁶ to 1%, 1×10⁻⁶ to 0.5%,1×10⁻⁶ to 0.1% or 1×10⁻⁶ to 0.01% w/w of the cosmetic composition if notfurther diluted after step (iv) or reduced by a factor of, e.g. 10-200,e.g. 30-100 if diluted in step (v).

The proportion of the polypeptides or portions of polypeptides derivablefrom fish hatching fluid in the cosmetic compositions may be definedrelative to the other solutes in the composition, i.e. excludingsolvents, e.g. water. Thus, said polypeptides or portions ofpolypeptides, in combination, may be present at the range of 1-100% w/wof the dry mass of the composition. In some embodiments the polypeptidesor portions of polypeptides, in combination, may be present at the rangeof 1-90% w/w of the dry mass of the composition, e.g. 5-80%, 10-70%,20-60%, 30-50% w/w of the dry mass of the composition. In otherembodiments the polypeptides or portions of polypeptides, incombination, may be present at the range of 1-40%, 2-39%, 3-38%, 4-37%etc. w/w of the dry mass of the composition. Thus, individualpolypeptides or portions of polypeptides may be present at the range of0.0001 to 50% w/w of the dry mass of the composition, e.g. 0.0001 to40%, 0.001 to 30%, 0.01 to 25% w/w of the dry mass of the composition.As described herein the composition may be diluted for use according tothe invention in step (v).

Whilst the invention is directed to methods for improving the cosmeticappearance of skin, this may include the treatment of a disorder,abnormality or condition, but in all cases the treatment is cosmetic innature.

As referred to herein “cosmetic” is intended to refer to a treatmentwhich does not cure, treat or prevent a disease or disorder, but insteadserves as a skincare product or to modify or improve the appearance ofthe skin, e.g. the colour, texture or moisture content of the skin.

The basis of the treatments described herein is the skin anti-ageingeffects of the cosmetic composition as disclosed herein. These effectshave been shown in the Examples provided herein.

Thus treatments based on the anti-ageing properties of the cosmeticcomposition are contemplated.

The invention thus provides a cosmetic method of improving theappearance of skin of a mammalian animal, wherein a cosmetic compositionas described hereinbefore is administered to said animal.

In a particularly preferred embodiment the skin is aged skin.

“Aged skin” refers to skin that displays one or more signs or symptomsof ageing, i.e. the appearance of wrinkles, fine lines,hyperpigmentation, laxity (sagging), dry skin, scaling or transepidermalwater loss (TEWL). In particular, “aged skin” is determined relative tonormal optimum skin, i.e. healthy, hydrated, normally pigmented andnon-aged skin. In this respect, aged skin need not be related to the ageof the subject and may be aged prematurely, e.g. by chronic exposure tosunlight (photo-damage). Thus, the relative parameters for “normaloptimum skin” may be determined as the average measurements of the abovesigns of ageing from a number of subjects of the same or similar age tothe subject in question, e.g. subjects that have not received chronicexposure to sunlight. Alternatively, the relative parameters for “normaloptimum skin” may be taken as the measurements from subjects that areyounger than the subject in question. In other words, the compositiondescribed herein may be used to restore the youthful appearance of skin,relative to the skin of the subject at an earlier age.

Thus, the invention provides a cosmetic method for the treatment ofdermatoheliosis in a mammalian animal wherein a cosmetic composition asdescribed hereinbefore is administered to said animal, preferablywherein said composition is administered topically.

Alternatively viewed, the invention provides a cosmetic composition asdescribed hereinbefore for use in the treatment of dermatoheliosis in amammalian animal, preferably wherein said cosmetic composition is foradministration to the skin of said animal. In a particular embodimentthe composition is for topical administration.

In a particularly preferred embodiment, improving the cosmeticappearance of skin (e.g. aged or photo-damaged skin) involves areduction or prevention in the cosmetic appearance or prevalence ofwrinkles, fine lines, hyperpigmentation, laxity, dry skin, scalingand/or transepidermal water loss. One or more of these parameters may beimproved. Preferably fine lines and/or wrinkles are reduced.

Reduction or prevention in the cosmetic appearance or prevalence of thesigns or symptoms of e.g. aged skin or dermatoheliosis, may mean thatthere is a reduction in the number and/or severity of the sign orsymptom. For instance, the number of fine lines and wrinkles may bereduced and/or the size, e.g. the depth, of the wrinkles or fine linesmay be reduced or minimized. Furthermore, reduction or prevention mayinvolve stopping, or reducing the rate of, the appearance of new signsor symptoms.

“Dry skin” as referred to herein refers to an epidermis that lacksmoisture or sebum, often characterized by a pattern of fine lines,scaling, and an itching and/or burning feeling. Dry skin can occur as askin condition in itself (e.g. due to age) or may be the symptom of askin disorder or condition such as sun-damage.

In this respect, the reduction of dry skin, scaling, fine-lines ortransepidermal water loss may be achieved by the moisturizing effects ofthe composition described above.

Thus, the invention may be seen to provide a cosmetic method ofmoisturizing skin of a mammalian animal, wherein a cosmetic compositionas defined herein is administered to said animal.

Alternatively stated, the present invention provides a cosmeticcomposition as described herein for use in moisturizing skin of amammalian animal. (The composition may alternatively be used to preparea cosmetic medicament for that purpose.)

“Moisturizing” as referred to herein covers moisturizers which preventloss of water from the skin (e.g. TEWL) as well as moisturizers(humectants) that attract and retain water when applied to the skin andemollients (which improve defective desquamation).

As mentioned above, such moisturizing properties are advantageous forimproving the cosmetic appearance of skin. In a particularly preferredembodiment, the skin is the skin of the face, ears, neck, hands orscalp.

“Wrinkles” are folds, ridges or creases in the skin. Skin wrinklestypically appear as a result of ageing processes. In this respect, thedermis comprises many of the structural elements of skin, which includecollagen, which gives the skin its strength, glycosaminoglycans whichgive the skin its turgor, and elastin fibres which give the skin itselasticity or spring.

As the skin ages, the dermal layer gets thinner and the skin alsoproduces less collagen. Moreover, the elastin fibres that provideelasticity wear out. These changes in the scaffolding of the skin causethe skin to wrinkle and sag. The rete-ridges of the dermal-epidermaljunction flatten out, making the skin more fragile and making it easierfor the skin to shear. This process also decreases the amount ofnutrients available to the epidermis by decreasing the surface area incontact with the dermis, also interfering with the skin's normal repairprocess.

In the subcutaneous layer the fat cells get smaller with age. This leadsto more noticeable wrinkles and sagging (laxity), as the fat cellscannot “fill in” the damage from the other layers.

Exposure to UVA and UVB radiation, i.e. sunlight, causes collagen tobreak down at a higher rate than with just chronologic ageing. Sunlightdamages collagen fibres and causes the accumulation of abnormal elastin.When this sun-induced elastin accumulates, matrix metalloproteinases(MMP) are produced in large quantities. Normally, metalloproteinasesremodel sun-injured skin by manufacturing and reforming collagen.However, this process does not always work well and some of themetalloproteinases actually break down collagen. This results in theformation of disorganized collagen fibres known as solar scars. Therepetition of this imperfect rebuilding/regeneration process causeswrinkles to develop and skin laxity.

In a further preferred aspect, the skin condition to be treated orprevented cosmetically is a pigmentation condition, disorder orabnormality.

Pigmentation disorders or abnormalities of the skin, i.e.hyperpigmentation, may occur as a result of age or may result frompremature ageing due to e.g. sun damage. Altered pigmentation may resultfrom a local excess of melanocytes or increases in melanocyte activity,or both. Pigmentation disorders include liver, sun or age spots (solarlentigo) and other blemishes such as freckles.

As referred to herein “improving” the cosmetic appearance of skin isdetermined relative to normal optimum skin, i.e. healthy, hydrated,normally pigmented and non-aged skin. Hence, with respect to aged skin,one or more of the signs or symptoms of ageing may be measured asdescribed in the Examples and compared to the same signs of skin that ischronologically or physiologically younger, preferably when animprovement is the reduction in one or more of the signs or symptoms ofageing.

In a preferred aspect the cosmetic uses are achieved by topicaladministration to the skin.

As used herein, “treating” refers to the reduction, alleviation orelimination, preferably to normal levels, of one or more of the cosmeticsymptoms or effects of said condition or disorder e.g. presence orextent of dry skin, extent or area of pigmentation etc. relative to thesymptoms or effects present on a different part of the body of saidindividual where the skin does not suffer from said condition ordisorder and not subject to said treatment or in a corresponding normalindividual not subject to said treatment.

“Preventing” or “reducing” refers to absolute prevention, or reductionor alleviation of the extent or timing (e.g. delaying) of the onset ofthat symptom or effect. For example conditions typified by dry,abnormally pigmented, wrinkled skin may be prevented by regularapplication of cosmetic compositions described herein before theappearance of such a condition.

The cosmetic methods of treatment or prevention according to theinvention may advantageously be combined with administration of one ormore active ingredients which are effective in treating or preventingthe disorders or conditions and/or to achieve, e.g. moisturization.Thus, cosmetic compositions described herein may additionally containone or more of such active ingredients.

According to a yet further aspect of the invention we providecompositions as herein defined and optionally one or more additionalactive ingredients as a combined preparation for simultaneous, separateor sequential use in human or mammalian animal therapy, as describedherein.

The compositions described herein may be formulated in a conventionalmanner with one or more physiologically acceptable carriers, excipientsand/or diluents, according to techniques well known in the art usingreadily available ingredients. The compositions may be provided as waterbased, glycerin based, alcohol (up to 20%) based, acrylate basedoil/water emulsions, xanthan gum based oil/water emulsions or water/oilemulsions, for example, at pH 3.5-11, preferably pH 5.5-9.

Thus, the compositions may be incorporated, optionally together withother active substances as a combined preparation, with one or moreconventional carriers, diluents and/or excipients, to produceconventional galenic preparations such as powders, sachets, cachets,elixirs, suspensions (infusion fluids), emulsions, solutions, syrups,aerosols (as a solid or in a liquid medium), ointments, sterile packagedpowders, and the like. The compositions may be stabilized by use offreeze-drying, undercooling or Permazyme. Such compositions formcompositions of the invention (i.e. are prepared in accordance with step(v)).

Suitable excipients, carriers or diluents are lactose, dextrose,sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate,calcium carbonate, calcium lactose, corn starch, aglinates, tragacanth,gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water syrup, water, water/ethanol,water/glycol, water/polyethylene, glycol, propylene glycol, methylcellulose, methylhydroxybenzoates, propyl hydroxybenzoates, talc,magnesium stearate, mineral oil or fatty substances such as hard fat orsuitable mixtures thereof. Agents for obtaining sustained releaseformulations, such as carboxypolymethylene, carboxymethyl cellulose,cellulose acetate phthalate, or polyvinylacetate may also be used.

The compositions may additionally include lubricating agents, wettingagents, emulsifying agents, viscosity increasing agents, granulatingagents, disintegrating agents, binding agents, osmotic active agents,suspending agents, preserving agents, sweetening agents, flavouringagents, adsorption enhancers (e.g. surface penetrating agents, e.g. bilesalts, lecithins, surfactants, fatty acids, chelators), browning agents,organic solvent, antioxidant, stabilizing agents, emollients, silicone,alpha-hydroxy acid, demulcent, anti-foaming agent, moisturizing agent,vitamin, fragrance, ionic or non-ionic thickeners, surfactants, filler,ionic or non-ionic thickener, sequestrant, polymer, propellant,alkalinizing or acidifying agent, opacifier, colouring agents and fattycompounds and the like. Some of these components are described in moredetail below.

Other active ingredients or components in the cosmetic composition maybe selected from any one or more of minerals, vitamins, enzymes,proteins, peptides, amino acids, lipids, polysaccharides, substancessuitable as sunscreen filters, chemical exfoliants, extracts,skin-conditioning agents, antioxidants and mixtures thereof.

Examples of proteins that may be included in the composition of theinvention include collagen and/or a derivative thereof (e.g. portionsthereof as defined above), a protein or peptide which is capable ofpromoting cell growth, glycoprotein 1, glycoprotein 2 and laminin.

The composition of the invention may be provided with enzymes including,but not limited to, any one or more of, fruit enzymes (e.g. bromelain),superoxide dismutase, peroxidase, hyaluronidase andmucopolysaccharidase.

Peptides may be selected from, but are not limited to, any one or moreof D,L-carnosine, D-carnosine, L-carnosine, anserine and Matrixyl(pentapetide derivative).

Amino acids may be selected from, but are not limited to, any one ormore of L-alanine, L-arginine, L-asparagine, L-aspartic acid,L-cysteine, L-cystine, glycine, L-glutamine, L-glutamic acid,L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine,L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan,L-tyrosine, and L-valine and derivatives thereof including non-naturallyoccurring amino acids as defined in Table 1. Particularly preferredamino acids as antioxidants may be selected from any one or more ofglycine, lysine, arginine, cysteine, cystine, histidine, tyrosine andtryptophan.

TABLE 1 Non-conventional Non-conventional amino acid Code amino acidCode α-aminobutyric acid Abu L-N-methylalanine Nmalaα-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmargaminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylateL-N-methylaspartic acid Nmasp aminoisobutyric acid AibL-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmglncarboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine ChexaL-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucineNmile D-alanine Dal L-N-methylleucine Nmleu D-arginine DargL-N-methyllysine Nmlys D-aspartic acid Dasp L-N-methylmethionine NmmetD-cysteine Dcys L-N-methylnorleucine Nmnle D-glutamine DglnL-N-methylnorvaline Nmnva D-glutamic acid Dglu L-N-methylornithine NmornD-histidine Dhis L-N-methylphenylalanine Nmphe D-isoleucine DileL-N-methylproline Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysineDlys L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophanNmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine DpheL-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine NmetgD-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine DthrL-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyrα-methyl-aminoisobutyrate Maib D-valine Dval α-methyl-γ-aminobutyrateMgabu D-α-methylalanine Dmala α-methylcyclohexylalanine MchexaD-α-methylarginine Dmarg α-methylcylcopentylalanine McpenD-α-methylasparagine Dmasn α-methyl-α-napthylalanine ManapD-α-methylaspartate Dmasp α-methylpenicillamine Mpen D-α-methylcysteineDmcys N-(4-aminobutyl)glycine Nglu D-α-methylglutamine DmglnN-(2-aminoethyl)glycine Naeg D-α-methylhistidine DmhisN-(3-aminopropyl)glycine Norn D-α-methylisoleucine DmileN-amino-α-methylbutyrate Nmaabu D-α-methylleucine Dmleu α-napthylalanineAnap D-α-methyllysine Dmlys N-benzylglycine Nphe D-α-methylmethionineDmmet N-(2-carbamylethyl)glycine Ngln D-α-methylornithine DmornN-(carbamylmethyl)glycine Nasn D-α-methylphenylalanine DmpheN-(2-carboxyethyl)glycine Nglu D-α-methylproline DmproN-(carboxymethyl)glycine Nasp D-α-methylserine Dmser N-cyclobutylglycineNcbut D-α-methylthreonine Dmthr N-cycloheptylglycine NchepD-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex D-α-methyltyrosineDmty N-cyclodecylglycine Ncdec D-α-methylvaline DmvalN-cylcododecylglycine Ncdod D-N-methylalanine Dnmala N-cyclooctylglycineNcoct D-N-methylarginine Dnmarg N-cyclopropylglycine NcproD-N-methylasparagine Dnmasn N-cycloundecylglycine NcundD-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine NbhmD-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine NbheD-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine NargD-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine NthrD-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine NserD-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine NhisD-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine NhtrpD-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate NmgabuN-methylcyclohexylalanine Nmchexa D-N-methylmethionine DnmmetD-N-methylornithine Dnmorn N-methylcyclopentylalanine NmcpenN-methylglycine Nala D-N-methylphenylalanine DnmpheN-methylaminoisobutyrate Nmaib D-N-methylproline DnmproN-(1-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nleu D-N-methylthreonine DnmthrD-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine NvalD-N-methyltyrosine Dnmtyr N-methyla-napthylalanine NmanapD-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acidGabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine TbugN-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine PenL-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine MargL-α-methylasparagine Masn L-α-methylaspartate MaspL-α-methyl-t-butylglycine Mtbug L-α-methylcysteine McysL-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamateMglu L-α-methylhistidine Mhis L-α-methylhomophenylalanine MhpheL-α-methylisoleucine Mile N-(2-methylthioethyl)glycine NmetL-α-methylleucine Mleu L-α-methyllysine Mlys L-α-methylmethionine MmetL-α-methylnorleucine Mnle L-α-methylnorvaline Mnva L-α-methylornithineMorn L-α-methylphenylalanine Mphe L-α-methylproline MproL-α-methylserine Mser L-α-methylthreonine Mthr L-α-methyltryptophan MtrpL-α-methyltyrosine Mtyr L-α-methylvaline MvalL-N-methylhomophenylalanine NmhpheN-(N-(2,2-diphenylethyl)carbamylmethyl)glycine NnbhmN-(N-(3,3-diphenylpropyl)carbamylmethyl)glycine Nnbhe1-carboxy-1-(2,2-diphenyl- Nmbc L-O-methyl serine Omserethylamino)cyclopropane L-O-methyl homoserine Omhser

The cosmetic composition may comprise one or more lipids which includesfats, oils, waxes and the like. Suitable polar oils are, for example,those from the group of lecithins and fatty acid triglycerides, namelythe triglycerol esters of saturated and/or unsaturated, branched and/orunbranched alkanecarboxylic acids with a chain length of from 8 to 24,in particular 12 to 18, carbon atoms. The fatty acid triglycerides can,for example, be chosen advantageously from the group of synthetic,semisynthetic and natural oils, such as, for example, olive oil,sunflower oil, soya oil, peanut oil, rapeseed oil, almond oil, palm oil,coconut oil, castor oil, wheat germ oil, grape seed oil, thistle oil,evening primrose oil, macadamia nut oil and the like.

Alternatively or additionally the oil may be selected from volatileoils, non-volatile oils or mixtures thereof. Non-volatile oils includeoils that fulfill at least one of the following definitions: (a) the oilexhibits a vapour pressure of no more than 0.2 mm Hg at 25° C. and oneatmosphere pressure; (b) the oil has a boiling point at one atmosphereof at least 300° C. Volatile oils include materials that are not“non-volatile” as defined above.

Non-volatile oils may be selected from non-volatile silicone oils,non-volatile hydrocarbon oils and mixtures thereof. Suitablenon-volatile silicone oils include linear polymethylsiloxanes and,preferably, non-volatile silicone oils are high molecular weightdimethicones. Examples of commercially available linearpolymethylsiloxanes include DC 200 Fluid 20 Cst, DC 200 Fluid 100 Cst,DC 200 Fluid 350 Cst from Dow Corning Corporation.

Suitable non-volatile hydrocarbon oils include branched esters ofdiglycerin or triglycerin or the esters or 1,2,3,4 butane triol orerythritol, di erythritol or tri erthyritol. Preferably, non-volatilehydrocarbon oils comprise erythrityl triethylhexanoate (available asSalacos E-38 from Nisshin Oilio) and Polyglyceryl-2 triisostearate(available as Cosmol 43V from Nisshin Oilio), diethyl hexyl carbonate(available as Tegosoft DEC from Degussa), dicapryl Ether (available asCetiol OE from Cognis AG), dicapryl Carbonate (available as Cetiol CCfrom Cognis AG), isononyl isononanoate (available as Lanol 99 fromSeppic), tridecyl Neopentanoate (supplied as Ceraphyl 55 fromInternational Speciality Products), or a mixture thereof.

Volatile oils may be selected from volatile silicone oils, bothfunctionalised and non-functionalised, volatile hydrocarbon oils andmixtures thereof. Volatile oil useful in the present invention may besaturated or unsaturated, have a straight or branched chain or a cyclicstructure or have any one or more of these features.

Examples of volatile hydrocarbons oils include polydecanes such asisododecane and isodecane (e.g., Permethyl-99A which is available fromPresperse Inc.) and the C7-C15 isoparaffins (such as the Isopar Seriesavailable from Exxon Chemicals).

The volatile silicone oil may be selected from cyclopentasiloxane,cyclohexasiloxane or a mixture thereof. Examples of commerciallyavailable volatile cyclic silicone oils include DC 244, DC 245, DC 344,and DC 345 from Dow Corning Corp.; SF-1204 and SF-1202 Silicone Fluidsfrom Momentive Performance Materials; GE 7207 and 7158 from GeneralElectric Co.); and, SWS-03314 from SWS Silicones Corp.

The linear volatile silicone oil may be a linear polymethylsiloxane. Anexample of commercially available linear polymethylsiloxanes includes DC200 Fluid, 5 Cst from Dow Corning Corp.

The cosmetic composition of the invention may further comprise one ormore polysaccharides selected from, but not limited to, any one or moreof anionic polysaccharides (e.g. alginic acid, pectin, xanthan gum,hyaluronic acid, chondroitin sulfate, gum arabic, gum karaya, gumtragacanth, carboxymethyl-chitin, cellulose gum, glycosaminoglycans),cationic polysaccharides (e.g. chitosan, acetylated chitosan, cationicguar gum, cationic hydroxyethylcellulose (HEC)), nonionicpolysaccharides (e.g. starch, dextrins, guar gum, cellulose ethers suchas hydroxyethylcellulose, methylcellulose and nitrocellulose),amphoteric polysaccharides (e.g. carboxymethylchitosan,N-hydroxy-dicarboxyethyl-chitosan, modified potato starch) andhydrophobic polysaccharides (e.g. cetyl hydroxyethylcellulose,polyquaternium24).

The cosmetic composition may further comprise a substance suitable as asunscreen filter such as an organic sunscreen, e.g. a cinnamicderivative. The organic sunscreen active may be selected fromhydrophilic organic sunscreen, hydrophobic organic sunscreen, ormixtures thereof. Suitable examples of sunscreens may be found in theCTFA International Cosmetic Ingredient Dictionary and Handbook, 7thedition volume 2, pp. 1672, edited by Wenning and Mc Ewen (The Cosmetic,Toiletry, and Fragrance Association, Inc., Washington, D.C. 1997).

The organic sunscreen may be selected from alkyl β,β-diphenylacrylatederivatives, α-cyano β,β-diphenylacrylate derivatives, anthranilatederivatives, benzophenone derivatives, camphor derivatives,dibenzoylmethane derivatives, p-aminobenzoic derivatives, salicylicderivatives, triazine derivatives, or mixtures thereof. For instance thehydrophobic organic sunscreen may be selected from4-(1,1-dimethylethyl)-4′-methoxydibenzoylmethane;4-isopropyldibenzoylmethane;4-(1,1-dimethylethyl)-4′-methoxydibenzoylmethane,2-ethylhexyl-2-cyano-3,3-diphenylacrylate, or a mixture thereof.

An example of commercially available4-(1,1-dimethylethyl)-4′-methoxydibenzoylmethane, also known as butylmethoxydibenzoylmethane or Avobenzone, includes Parsol TM 1789 fromGivaudan Roure S. A. and Eusolex TM 9020 from Merck & Co., Inc. Anexample of commercially available 4-isoproplydibenzoylmethane, alsoknown as isopropyldibenzoylmethane, includes Eusolex TM 8020 from Merck& Co., Inc. Examples of commercially available2-ethylhexyl-2-cyano-3,3-diphenylacrylate, also known as Octocrylene,include Uvinul N539 SG from BASF; and Eusolex OCR from Rona/Merck.

In some embodiments the hydrophilic organic sunscreen may be2-phenylbenzimidaole-5-sulfonic acid. An example of commerciallyavailable 2-phenylbenzimidaole-5-sulfonic acid, also known as PBSA,includes Eusolex 232 from Rona/Merck.

Suitable examples of cinnamic derivative sunscreens may be found in theCTFA International Cosmetic Ingredient Dictionary and Handbook, 7thedition volume 2, pp. 1672, edited by Wenning and Mc Ewen (The Cosmetic,Toiletry, and Fragrance Association, Inc., Washington, D.C. 1997). Thecinnamic derivative may be selected from2-ethylhexyl-p-methoxycinnamate, diethanolamine methoxycinnamate,2-ethoxyethyl-p-methoxycinnamate, or a mixture thereof. For instance,the cinnamic derivative may be 2-ethylhexyl-p-methoxycinnamate.

The cosmetic composition may contain a chemical exfoliant selected from,but not limited to, any one more of alpha hydroxy acids (AHAs), betahydroxy acids (BHAs) or poly-hydroxy acids, such as salicylic acid,glycolic acid, citric acid and malic acid.

Extracts that may be incorporated in the cosmetic composition include,but are not limited to plant extracts, which may comprise phenoliccompounds such as, for example, flavonoids (e.g., glycosyl rutin,ferulic acid, caffeic acid), furfurylidene glucitol, butylatedhydroxytoluene, butylated hydroxyanisole, nordihydroguaiaretic resinacid, nordihydroguaiaretic acid, trihydroxybutyrophenone and derivativesthereof. Particular plant extracts for use in the composition of theinvention include aloe vera extract, ginseng extract and horsetailextract.

Ginseng extract is obtainable by extracting with a hydrophilic solvent(in particular, water, ethanol, glycol, or any mixtures thereof) theroot of Panax ginseng. The extract contains saponins, sterols,carbohydrates, pectin, vitamins, minerals and lipids.

Horsetail extract is obtainable by extracting with a hydrophilic solvent(e.g., water, ethanol, glycol, or any mixtures thereof) the whole herbof Equisetum arvense. The extract contains silicates, flavinoids,saponosides, caffeic acid and ferulic acid.

The cosmetic composition may further comprise a skin-conditioning agent.The skin-conditioning agent may be selected from humectants, exfoliants,emollients or mixtures thereof. Humectants includes polyhydric alcoholssuch as glycerine, propylene glycol, dipropylene glycol, polypropyleneglycol, polyethylene glycol, sorbitol, hydroxypropyl sorbitol, hexyleneglycol, 1,3-butylene glycol, 1,2,6-hexanetriol, ethoxylated glycerin,propoxylated glycerine or mixtures thereof.

Examples of antioxidants that may be provided in the composition of theinvention include but are not limited to amino acids, vitamins,minerals, carotenoids, peptides, thiols, sulfoximine compounds,chelators, unsaturated fatty acids, phenolic compounds, plant extracts,stilbenes, uric acid, mannose, chlorogenic acid, imidazoles (e.g.urocanic acid), furfurylidenesorbitol, ubiquinone, ubiquinol,plastoquinone, phytosterols and derivatives thereof (e.g. salts, esters,ethers, sugars, nucleotides, nucleosides, peptides and/or lipidderivatives), some of which are described above.

Vitamins may be selected from, but are not limited to, any one or moreof vitamin A and derivatives thereof (e.g. retinoid or retinol or theirderivatives such as retinyl palmitate or retinyl proprionate), biotin,folic acid, calcium pantothenate, nicotinamide, pyridoxine HCl,pyridoxal HCl, riboflavin, thiamine HCl, thymidine, vitamin B12, vitaminB3 (e.g. niacinamide), vitamin B5 (e.g. panthenol), vitamin C andderivatives thereof (e.g., ascorbyl palmitate, Mg ascorbyl phosphate,ascorbyl acetate), tocopherols and derivatives (e.g. vitamin E acetate).

Minerals may be selected from, but are not limited to, any one or moresalts of molybdenate (e.g. (NH₄)OMo₇O₂₄) aluminium (e.g. AlCl₃), calcium(e.g. CaCl₂), cobalt (e.g. CoCl₂), chromium (e.g. CrK(SO₄)), copper(e.g. CuSO₄), iron (e.g. Fe(NO₃)₃, FeSO₄), potassium (e.g. KCl),magnesium (e.g. MgCl₂), manganese (e.g. MnCl₂, MnSO₄), phosphate (e.g.Na₂HPO₄, NaH₂PO₄), carbonate (e.g. NaHCO₃), silicate (e.g. Na₂SiO₃),sodium (e.g. NaCl), vanadate (e.g. NH₄VO₃), nickel (e.g. NiCl₂), tin(e.g. SnCl₂), zinc (e.g., ZnO, ZnSO₄), selenium (e.g. selenomethionine,ebselen, H₂SeO₃, Na₂SeO₃), sulphate and nitrate.

Carotenoids, may be selected from, but are not limited to, any one ormore of carotenes, e.g. α-carotene, β-carotene, ψ-lycopene, phytoeneetc. and derivatives thereof.

Thiols may be selected from, but are not limited to, any one or more ofaurothioglucose, propylthiouracil, thioredoxin, lipoic acid,glutathione, cysteine, cystine, cystamine and their glycosyl, N-acetyl,methyl, ethyl, propyl, amyl, butyl and lauryl, palmitoyl, oleyl,γ-linoleyl, cholesteryl and glyceryl esters and the salts thereof,dilauryl thiodipropionate, distearyl thiodipropionate, thiodipropionicacid and derivatives thereof.

Sulfoximine compounds may be selected from, but are not limited to, anyone or more of homocysteine sulfoximine, buthionine sulfones, penta-,hexa-, heptathionine sulfoximine, which may be included in thecomposition such that they are provided in very low dosages (e.g. pmolto μmol/kg).

Chelators may be selected from, but are not limited to, any one or moreof apoferritin, desferral, lactoferrin, α-hydroxy fatty acids, palmiticacid, phytic acid, α-hydroxy acids (e.g. citric acid, lactic acid, malicacid), humic acid, bile acid, bile extracts, bilirubin, biliverdin,EDTA, EGTA and derivatives thereof.

Unsaturated fatty acids may be selected from, but are not limited to,any one or more of γ-linolenic acid, linoleic acid, oleic acid andderivatives thereof.

Stilbenes and derivatives thereof include, for example, stilbene oxideand trans-stilbene oxide.

A variety of additional optional active ingredients may be incorporatedinto the cosmetic compositions of the present invention. Non-limitingexamples of these additional ingredients include additional skin careactives such as farnesol, bisabolol, phytantriol, urea, guanidine (e.g.amino guanidine); hexaminidine compounds, salts or derivatives thereof;sugar amines; self-tanning agents (e.g. dehydroxyacetone); structuringagents; hydrophilic gelling agents; anti-acne medicaments (resorcinol,salicylic acid, and the like); skin soothing and healing agents such asallantoin and the like; and agents suitable for aesthetic purposes suchas essential oils, fragrances, skin sensates, opacifiers, aromaticcompounds (e.g. clove oil, menthol, camphor, eucalyptus oil, andeugenol). The compositions described herein may be formulated so as toprovide quick, sustained or delayed release of the active ingredientsafter administration to the body by employing techniques well known inthe art.

The composition may be in any appropriate dosage form to allow deliveryor for targeting particular cells or tissues, e.g. as an emulsion or inliposomes, niosomes, microspheres, nanoparticles or the like with whichthe active ingredient may be absorbed, adsorbed, incorporated or bound.This can effectively convert the product to an insoluble form. Theseparticulate forms may overcome both stability (e.g. degradation) anddelivery problems.

The use of solutions, suspensions, gels and emulsions are preferred,e.g. the active ingredient may be carried in water, a gas, a water-basedliquid, an oil, a gel, an emulsion, an oil-in water or water-in-oilemulsion, a dispersion or a mixture thereof.

The emulsifier may be selected from nonionic emulsifiers, anionicemulsifiers, cationic emulsifiers, zwitterionic emulsifiers, amphotericemulsifiers or mixtures thereof. Emulsifiers are known in the art. See,e.g., McCutcheon's, Detergents and Emulsifiers, North American Edition(1986), published by Allured Publishing Corporation.

When the cosmetically acceptable carrier is a water-in-siliconeemulsion, emulsifiers are preferably selected from polyoxyalkylenecopolymers, polyglyceryl copolymers or mixtures thereof. Polyoxyalkylenecopolymers, also known as silicone polyethers, are described in detailin U.S. Pat. No. 4,268,499. An example of commercially availablepolyoxyalkylene copolymers includes DC5225C or DC2-5185C (PEG/PPG-18/18dimethicone available as blend with cyclopentasiloxane) from Dow CorningCorp.; and, KF6017 or KF6028 (PEG-9 dimethicone) from Shin-Etsu Inc.Examples of commercially available polyglyceryl emulsifiers includeKF6100 and KF6104 from Shin-Etsu Inc.

Compositions are preferably for topical (i.e. to the skin)administration.

Topical compositions include gels, creams, ointments, sprays, lotions,salves, sticks, soaps, powders, films, aerosols, drops, foams,solutions, emulsions, suspensions, dispersions e.g. non-ionic vesicledispersions, milks and any other conventional cosmetic forms in the art.

Ointments, gels and creams may, for example, be formulated with anaqueous or oily base with the addition of suitable thickening and/orgelling agents. Lotions may be formulated with an aqueous or oily baseand will, in general, also contain one or more emulsifying, dispersing,suspending, thickening or colouring agents. Powders may be formed withthe aid of any suitable powder base. Drops and solutions may beformulated with an aqueous or non-aqueous base also comprising one ormore dispersing, solubilising or suspending agents. Aerosol sprays areconveniently delivered from pressurised packs, with the use of asuitable propellant.

In some embodiments the cosmetic compositions described herein may betopically administered to the skin via a product, device or material towhich the polypeptide or composition has been applied, impregnated orchemically bonded. To this end, bandages, plasters (e.g. adhesivepatches), gauze, surgical tape, cotton swabs or other absorbentmaterials, e.g. a puff, fleece, or sponge, or supportive matrices may becoated, impregnated or chemically bonded with a composition as describedherein. For example, many compositions can be applied to the skin usingdermal patches that are well described in the art, e.g. US 2008/0038300,US 2009/0043236, WO 2005/067499 and WO 2009/085302, which areincorporated herein by reference. In some embodiments, the materialcomprising the composition as described herein may be in the form of adevice that can be, e.g. worn by the subject to be treated. Forinstance, the composition as described herein may be applied,impregnated or chemically bonded onto a material or supportive matrixthat forms all or part of a diaper, glove, sock etc.

The cosmetic compositions can be included in a container, pack, ordispenser together with instructions for administration.

Hence, a further aspect of the invention comprises the provision of aproduct, material or device which is coated, impregnated or chemicallybonded with a composition as described herein. The invention alsoextends to such products, materials or devices for uses as describedherein. Preferably said product is a bandage, plaster (e.g. adhesivepatch), gauze, surgical tape or cotton swab or said device is a diaper,glove or sock.

The concentration of the active ingredients in compositions describedherein, may depend upon the source of the composition (i.e. the startingmaterial for the method described above), the mode of administration,the course of treatment, the age and weight of the patient, the cosmeticindication, the body or body area to be treated and may be varied oradjusted according to choice. Generally however, the compositionprepared according to the method of the invention after step (iv) isdiluted in step (v) to 0.001, 0.005, 0.01 or 0.1 to 50%, e.g. 0.005-40%,e.g. 0.1 to 25%, such as 0.1 or 0.5 to 5, e.g. 1-5% (w/w or v/v) toprovide the final preparation for administration, particularly fortopical administration, e.g. a 1% or 3% solution of the compositionafter step (iv).

When additional components are added to the composition made by theabove described method, e.g. additional moisturizing agents as describedherein, the additional component may be present in the amounts 0.0001,0.0005, 0.001 or 0.01 to 50%, e.g. 0.0005-40%, e.g. 0.01 to 25%, such as0.1 or 0.5 to 5, e.g. 1-5% (w/w of the final preparation foradministration, particularly for topical administration). Effectivesingle doses for the composition may lie in the range of from 0.0001-100mg/cm²/day (total protein in the composition), e.g. 0.1-100 mg/cm²/day,preferably 0.0001-10 mg/cm²/day, e.g. 0.1-10 mg/cm²/day, when appliedtopically, depending on the mammalian animal being treated, taken as asingle dose.

Preferably liquid solutions, creams or suspensions would be employed fortopical administration.

Animals to which the compositions may be applied or administered arelimited to mammals. Preferably the mammals are primates, domesticanimals, livestock and laboratory animals. Thus preferred mammaliananimals include mice, rats, rabbits, guinea pigs, cats, dogs, monkeys,pigs, cows, goats, sheep and horses. Especially preferably thecompositions are applied, or administered, to humans.

The following Examples are given by way of illustration only in whichthe Figures referred to are as follows:

FIG. 1 shows a photograph of a subject treated with the hatching fluidcomposition of the invention before treatment (Baseline), after 2 weeksand after 12 weeks of treatment. The reduction in various signs of agedskin are evident after both 2 and 12 weeks. The values provided indicatethe average changes for 35 participants.

FIG. 2 shows a close-up photograph of the subject in FIG. 1 to emphasisethe reduction of fine lines and wrinkles seen after 2 and 12 weeks oftreatment with the hatching fluid composition of the invention.

FIG. 3 shows a close-up photograph of a subject treated with thehatching fluid composition of the invention before treatment (Baseline)and after 12 weeks of treatment. The circled area shows a clearreduction of wrinkles after 12 weeks of treatment.

FIG. 4 shows a bar chart depicting the percentage of subjects that wereconsidered to have improved in various signs of ageing based on atactile/visual clinical grading on both sides of the face.

FIG. 5 shows scanning electronmicrographs (×400, ×5000 and ×15000magnification) of reconstructed human epidermis treated with water, 5%glycolic acid, 1 mU/ml Bromelain or 1% hatching fluid composition for 12hours.

FIG. 6 shows scanning electronmicrographs (×400, ×5000 and ×15000magnification) of reconstructed human epidermis treated with water, 5%glycolic acid, 1 mU/ml Bromelain or 1% hatching fluid composition for 48hours.

FIG. 7 shows light micrographs of sections of reconstructed humanepidermis untreated or treated with 5% glycolic acid, 1 mU/ml Bromelainor 1% hatching fluid composition for 12 or 24 hours. Arrow (A) showscell proliferation and differentiation and arrow (B) shows denserstratum granulosum and a higher concentration of lamellar granules.

FIG. 8 shows (A) a SDS-PAGE gel stained with Coomassie and (B) a Westernblot of the gel in (A) stained with an antibody raised against aprinciple component of a cosmetic composition from derived from Salmonhatching fluid. Lane 1, Salmon hatching fluid; lane 2, Medaka hatchingfluid; and lane 3, Zebrafish hatching fluid.

EXAMPLE 1: PREPARATION OF THE COMPOSITION

The composition was prepared from salmon hatching fluid. To improve theprotein concentration of hatching fluid, salmon eggs were transferred tominimal volumes of water prior to hatching. Highly synchronous hatchingcan be induced by elevated (room) temperatures, or by deoxygenation(Oppen-Berntsen et al. 1990, Aquaculture, 86, pp. 417-430), which yieldsa small volume of highly concentrated preparation of crude polypeptidesand portions of polypeptides. Hatching should be complete within 2 hoursfor more than 95% of the embryos.

The hatching fluid was filtered using a standard filter with a 7 μm poresize, to remove material likely to clog filters in subsequent filtrationsteps. This filtrate, the processed hatching fluid, may be frozen foryears without significant degradation, before being thawed and employedfor further protein purification. This fact greatly simplifiesproduction of a starting material for purifying the hatching fluidcomposition.

The processed hatching fluid was subjected to filtration using a filterwith a 0.45 μm pore size and the filtrate was collected. The filtratewas then dia-filtrated with a filter exclusion size of 8 kDa to exchangewater of hatching fluid for buffer. In this case, the buffer contained0.5 mM phosphate and 1 mM NaCl, although other buffers are equallysuitable. For example, phosphate buffered saline or buffers containingmillimolar Tris (e.g. 10 mM) at pH around neutrality or slightlyalkaline (pH 7.5-8.5), containing 5 mM NaCl, are suitable. The retentatefrom the diafiltration step was collected and diluted by the addition ofthe buffer so that the enzymatic activity of the filtrate, as definedabove, was equal to 3400 mU/liter.

Finally, the filtrate was subjected to filtration through a filter witha pore size of 0.22 μm and the final filtrate was collected. Thisfiltrate is an enriched preparation of the polypeptides and portions ofpolypeptides, found in the crude hatching fluid.

EXAMPLE 2: IN VIVO EFFECTS OF HATCHING FLUID COMPOSITION ON AGED SKIN

The hatching fluid composition was prepared as described in Example 1.The composition was prepared as a 1% and 3% skin lotion [v/v] (totalvolume of composition per unit volume of lotion), the two active skinlotions in the trial, and compared to a control skin lotion which didnot comprise the active component, i.e. the hatching fluid composition.The skin lotion was an oil in water (O/W) emulsion. The oil phaserepresents 9% of the total composition and was emulsified withhydrogenated lecithin.

A double blind, placebo controlled clinical trial was conducted toevaluate the effectiveness and tolerance of topical skin treatments infemales with mild to moderate photodamaged, i.e. aged, facial skin. Theduration of this trial was 12 weeks with visits at baseline, Week 2,Week 6 and Week 12. Efficacy was assessed using visual grading,instrumentation, digital VISIA CR photographs and subjectself-assessment questionnaires.

Number of Subjects

One hundred and one (101) female subjects completed participation in thestudy (N>30 for the three treatments, i.e. one placebo and twocompositions comprising the hatching fluid component at differentconcentrations).

Subject Population and Identification

Subjects were healthy females ages 40 to 65 and were assigned athree-digit number which, when used in conjunction with the clinicalstudy number, uniquely identified every subject in the study. Thisnumber remained with the subject throughout the study to maintain theanonymity of the experiment.

Eligibility Criteria

Inclusion Criteria

1. Females, ages 40 to 65, inclusive, who were in general good health asdetermined by the health and eligibility questionnaire.

2. Willingness to cooperate and participate by following studyrequirements for the duration of the study and to report any adversesymptoms immediately.

3. Clinically determined mild to moderate photodamage (fine lines,wrinkles, hyperpigmentation, laxity and roughness) on the facecorresponding to the modified Griffith's grading scale with scores of3-7.

4. Free of any disease state or physical facial skin conditions (e.g.atopic dermatitis, eczema, psoriasis, seborrheic dermatitis) which mightimpair evaluations of the test sites or increase the health risk to thesubject by study participation.

5. Willingness to avoid extended periods of sun exposure and all use oftanning beds for the duration of the study. Extra care should be takento wear protective clothing, including sunglasses, and avoid sunexposure from 10 AM to 4 PM.

6. Willingness to continue use of all regular brands of colourcosmetics, cleanser, toner (if applicable) and makeup remover for theduration of the study. Individuals had to refrain from using anyanti-ageing products or skin lightening products other than the assignedtest material.

7. Willingness to remove all makeup at least 20 minutes prior to eachscheduled clinic visit. No other topical products were to be applied tothe face or eye area until the study visit was completed. If a subjectarrived having not removed all makeup, she was required to remove theresidual makeup at the clinic and wait at least 20 minutes prior toprocedures.

8. Individuals who were taking hormone replacement therapies or hormonesfor birth control had to be on a stable regimen for at least one monthprior to the study start and they had to be willing to continue and notchange this medication for the duration of the study. Individuals whowere not taking HRT or hormones at the start of the study had to bewilling to not begin use during the course of the study.

9. Willingness to cooperate and participate by following studyrequirements and to report any adverse symptoms immediately.

Exclusion Criteria

1. Individuals with a history of intolerance or allergy to any personalcare product.

2. Individuals who had used any prescription or OTC skin lighteningproducts less than 30 days prior to the study entry.

3. Individuals who had a condition and/or disease of the skin that theexamining Investigator deemed inappropriate for participation.

4. Individuals who were nursing, pregnant, or planning to becomepregnant during the study.

5. Individuals who had routinely used any anti-ageing, anti-wrinkle,topical antioxidants, less than 30 days prior to the study entry.

6. Individuals who had used an enzymatic facial skin treatment within 6months of the study start.

7. Use of Retin-A®, Retin-A Micro®, Renova®, Avita®, Tazorac®, Avage® orDifferin® or other topical retinoids within 3 months of the study start,or had taken Accutane or an oral retinoid within the past 6 months.

8. Routine use of products containing alpha-, beta- or poly-hydroxyacid(including salicylic acid and Lachydrin), retinol or derivatives ofretinol or other ‘anti-ageing’ products on the face within 30 days ofthe study start.

9. Individuals who had received a facial dermabrasion or chemical peeltreatment within 3 months of treatment or during the study.

10. Individuals who had received treatment with light RF, or otherdevices in the treated area within the treated area within 6 months oftreatment or during the study.

11. Individuals who had received Botox, collagen, fat injections orother methods of augmentation with injected or implanted material in thetreated area within 9 months of treatment or during the study.

12. Individuals who had undergone a resurfacing procedure, face lift oreye or eyelid surgery within 12 months prior to the start of this trial.

13. Individuals who had pre-existing and/or dormant dermatologicconditions on the face (e.g., vitiligo, atopic dermatitis, psoriasis,rosacea, eczema, seborrheic dermatitis, severe excoriations etc.) ormedical condition/disease which in the opinion of the Investigator couldhave interfered with the outcome of the study.

14. Individuals who had a history of immunosuppressant/immune deficiencydisorders (including (HIV infection or AIDS) or currently usingimmunosuppressive medications.

15. Individuals who were participating in any other clinical usage study(patch studies are acceptable).

16. Individuals who had an uncontrolled disease such as diabetes,hypertension, hyperthyroidism or hypothyroidism. Some individuals whohad multiple health conditions were excluded from participation even ifthe conditions are controlled by diet, medication, etc.

17. Individuals who had participated in any clinical trial within 28days prior to inclusion into the study.

Individuals were admitted to the study at the discretion of theInvestigator or his designate based on medical history and findings ofthe pre-study interview and examination.

Study Design

The double blind, placebo controlled clinical trial was conducted toevaluate the effectiveness of topical skin treatments in females withmild to moderate photodamaged, i.e. aged, facial skin. The duration ofthis trial was 12 weeks with visits scheduled at baseline, Week 2, Week6 and Week 12. Efficacy was assessed using visual grading,instrumentation, digital VISIA CR photographs and subjectself-assessment questionnaires.

Three groups of N>30 per group completed the study. Subjects received anactive skin treatment, namely the hatching fluid composition describedabove, or a vehicle control (water) to apply to the face for twelveweeks. Randomization of subjects into the 3 groups was performedaccording to a pre-determined randomization.

Visit: Visit 1 Visit 2 Visit 2 Visit 2 Baseline Week 2 Week 6 Week 12Informed Consent, eligibility paperwork, facial screening X Right andleft side clinical scoring for lines, wrinkles, mottled X X X Xhyperpigmentation, laxity, clarity and roughness Right and left sideclinical scoring for objective and subjective X X X X irritation(erythema, dryness, burning/stinging*, itching*, tight/dry feeling*) *reported by the panelist. Right and left side VISIA-CR imaging X X X XCutometer measurements on the right and left face. X X X XTransepidermal water loss (TEWL) measurements on the right X X X X andleft face. Distribution of test material, vehicle, usage instructions,diary X X and calendar Completion of self assessment questionnaires forright and left X X X face. Diary review and product weighing forcompliance X X X

Efficacy and Tolerability Evaluations

An expert clinical grader assessed the right and left side of the facefor the parameters shown below. A modified Griffith's scale was used,where 0=none, 1-3=mild, 4-6=moderate and 7-9=severe. Half points wereused when needed to better describe the skin condition.

-   -   Fine Lines    -   Wrinkles    -   Hyperpigmentation    -   Laxity    -   Dull/Matte (Clarity)    -   Tactile Roughness

An expert clinical grader assessed the right and left side of the facefor the parameters shown below. A four point scale was used, where0=none, 2=mild, 3=moderate and 4=severe. Half points were used whenneeded to better describe the skin condition.

-   -   Erythema    -   Dryness/scaling    -   Burning/stinging feeling    -   Itching    -   Tight/dry feeling

Digital VISIA CR Photography

VISIA-CR imaging was taken of the right and left sides of the face. Thesubjects were imaged such that their hair was pulled back, jewelry wasremoved, eyes were closed, the subject was centered within the frame andhad a neutral facial expression.

Transepidermal Water Loss (TEWL)

Prior to Instrumental measurements, subjects were made to equilibrate toambient conditions of the clinic for at least 20 minutes. Ambientconditions were recorded hourly during the study visits. During thistime, subjects were graded, completed questionnaires and/or had VISIA CRimaging performed.

The Tewameter was used to take a transepidermal water loss (TEWL)measurement at all visits. Measurements were taken on the right and leftcheek at the intersection of lines extending down from the corner of theeye and horizontally across the bottom of the nose.

The Tewameter measures TEWL utilizing an open chamber system. A handheld probe placed on the skin surface sampled relative humidity at twopoints above the surface, allowing the rate of water loss to becalculated from the measured humidity gradient.

Cutometer MPA 580

All subjects had Cutometer measurements taken at all visits. TheCutometer was used to assess the viscoelastic properties (i.e.extensibility and elasticity) of the skin. The instrument applies avacuum to a small area of skin and measures the elastic response of theskin (movement of the skin into and out of the aperture) by an opticaltechnique.

For this study, the 2 mm probe was used, a vacuum of 300 mbar wasapplied and two cycles of suction and release were performed. Cycletimes was 5 seconds on and 10 seconds off.

Measurements were taken on the right and left cheek at the intersectionof lines extending down from the corner of the eye and horizontallyacross the bottom of the nose, or an alternate location near the jaw.

Skin Assessment and Self-Assessment Questionnaires

Subjects completed a skin self assessment questionnaire containingquestions that describe how the subject perceives their facial skinappearance and condition on the right and left sides of the face.

Results

Fine Lines

At week 2 subjects treated with a skin lotion comprising one of theactive compositions showed a reduction in fine lines (e.g. percentagechange of 5.59% (1% solution) and 5.65% (3% solution)) in comparison tothe placebo (4.58%). The reduction in fine lines continued at week 6(e.g. 14.34% (1% solution), 14.86% (3% solution) and 8.98% (placebo))and week 12 (e.g. 23.43% (1% solution), 25.99% (3% solution) and 14.68%(placebo)). FIGS. 1 and 2 show a subject with a 28% reduction of finelines.

Wrinkles

At week 2 subjects treated with a skin lotion comprising one of theactive compositions showed a reduction in wrinkles (e.g. percentagechange of 2.15% (1% solution) and 1.75% (3% solution)) in comparison tothe placebo (0.70%). The reduction in wrinkles continued at week 6 (e.g.6.13% (1% solution), 7.32% (3% solution) and 3.70% (placebo)) and week12 (e.g. 14.72% (1% solution), 15.15% (3% solution) and 9.57%(placebo)). FIG. 1 shows a subject with a 12.5% reduction in wrinkles.FIG. 3 shows a subject with a 26.32% reduction in wrinkles.

Hyperpigmentation

At week 2 subjects treated with a skin lotion comprising one of theactive compositions showed a reduction in hyperpigmentation (e.g.percentage change of 2.11% (1% solution) and 2.68% (3% solution)) incomparison to the placebo (0.40%). The reduction in hyperpigmentationcontinued at week 6 (e.g. 5.61% (1% solution), 7.91% (3% solution) and3.16% (placebo)) and week 12 (e.g. 10.53% (1% solution), 15.35% (3%solution) and 5.73% (placebo)). FIG. 1 shows a subject with an 15%reduction in the pigmentation of an age spot after 12 weeks.

Laxity

At week 2 subjects treated with a skin lotion comprising one of theactive compositions showed a reduction in laxity (e.g. percentage changeof 2.64% (1% solution) and 1.62% (3% solution)) in comparison to theplacebo (0.87%). The reduction in laxity continued at week 6 (e.g. 6.33%(1% solution) and 6.61% (3% solution), 2.51% (placebo)) and week 12(e.g. 10.55% (1% solution) and 11.33% (3% solution), 5.18% (placebo)).FIG. 1 shows a subject with a 7.69% reduction in laxity (sagging) after12 weeks.

Dull/Matte (Clarity)

At week 2 subjects treated with a skin lotion comprising one of theactive compositions showed an improvement in skin clarity (e.g.percentage change of 12.95% (1% solution) and 16.00% (3% solution)) incomparison to the placebo (10.67%). The improvement continued at week 6(e.g. 29.26% (1% solution), 28.50% (3% solution) and 19.07% (placebo))and week 12 (e.g. 37.17% (1% solution), 39.18% (3% solution) and 26.72%(placebo)). FIG. 1 shows a subject with a 33.33% reduction in dullness.

Tactile Roughness

At week 2 subjects treated with a skin lotion comprising one of theactive compositions showed a reduction in the tactile roughness of theskin (e.g. percentage change of 16.51% (1% solution) and 20.24% (3%solution)) in comparison to the placebo (13.38%). The improvementcontinued at week 6 (e.g. 24.77% (1% solution), 26.65% (3% solution) and16.79% (placebo)), but there was not a further reduction at week 12(e.g. 26.61% (1% solution), 29.19% (3% solution), and 15.79% (placebo)).FIG. 1 shows a subject with a 12.5% reduction in tactile roughness.

Dryness/Scaling

At week 2 subjects treated with a skin lotion comprising one of theactive compositions showed a reduction in the dryness/scaling of theskin (e.g. percentage change of 72.09% (1% solution) and 100.00% (3%solution)) in comparison to the placebo (64.71%). However, at week 6(e.g. 86.05% (1% solution), 84.62% (3% solution) and 100.00% (placebo)),and week 12 there was not a further reduction when compared to theplacebo (e.g. 90.70% (1% solution), 100.00% (3% solution) and 89.47%(placebo)).

TEWL

Whilst subjects treated with a skin lotion comprising one of the activecompositions showed a reduction in TEWL at week 2, this was not clearlydifferent to the placebo (e.g. percentage change of 15.35% (1% solution)and 14.53% (3% solution)) in comparison to the placebo (17.26%).However, at week 6 (e.g. 29.46% (1% solution), 26.66% (3% solution) and22.96% (placebo)), and week 12 there was a further reduction greaterthan that of the placebo (e.g. 37.46% (1% solution), 40.04% (3%solution), and 34.21% (placebo)).

Extensibility

Whilst subjects treated with a skin lotion comprising one of the activecompositions showed an improvement in the extensibility of the skin atweek 2, this was only slightly different to the placebo (e.g. percentagechange of 16.18% (1% solution) and 17.21% (3% solution)) in comparisonto the placebo (10.82%). At week 6 there was no clear difference betweenthe three treatments (e.g. 18.04% (1% solution), 17.18% (3% solution)and 19.90% (placebo)), but at week 12 there was a further improvementfor the skin treatment with the compositions comprising the activecomponent, which was greater than that of the placebo (e.g. 31.84% (1%solution), 33.57% (3% solution), and 16.48% (placebo)).

A comparison of the tactile/visual clinical grading on both sides of theface performed at the start of the study (baseline) and after 12 weeksof treatment shows that the all of the subjects showed improvements inthe dullness and roughness of their skin following treatment with thehatching fluid composition of the invention and the majority of subjectshowed improvements in fine lines (97% of subjects), wrinkles (91% ofsubjects), hyperpigmentation (87% of subjects) and sagging (80% ofsubjects) (FIG. 4).

Questionnaires reveal that from 6 weeks of use statistically significantdifferences were found for mean scores of statements about overallappearance, overall feel, smoothness, softness, clarity and elasticitybetween the placebo and the cosmetic composition comprising the hatchingfluid composition of the invention.

Thus, it is evident from the above results that the hatching fluidextract composition demonstrated an effect on each aspect of aged skinin comparison to the placebo.

EXAMPLE 3: COMPARATIVE IN VITRO STUDY OF EFFECTS OF HATCHING FLUIDCOMPOSITION ON RECONSTRUCTED HUMAN EPIDERMIS RELATIVE TO KNOWN COSMETICSKIN TREATMENTS

In vitro reconstructed human epidermis consists of normal humankeratinocytes cultured on an inert polycarbonate filter at theair-liquid interface, in a chemically defined medium. This in vitromodel is histologically similar to that of the in vivo human epidermis.

Reconstructed Human Epidermis (SkinEthic) was exposed on the stratumcorneum side for 12 hours, 24 hours or 48 hours to 200 μl of one of thefollowing solutions:

1% Hatching fluid composition;

5% Glycolic Acid (AHA);

1 mU/ml Bromelain (fruit enzyme); or

dH₂O (control).

After exposure the cultures were fixed in 4% (w/v) paraformaldehyde inPBS at 4° C. and analysed by scanning electron or light microscopy.

Evalution of scanning electronmicrographs of the treated reconstitutedhuman epidermis after 12 hours (FIG. 5) shows that treatment with a 5%solution of glycolic acid results in damaged stratum corneum and skincorrosion. Treatment with Bromelain results in desquamation by digestionof the cells' envelope. In contrast, treatment with the hatching fluidcomposition of the invention results in desquamation by shedding ofintact corneocytes because only the cell binding sites have beendegraded. Thus, the hatching fluid solution provides gentlemicro-exfoliation of the skin.

After exposure for 48 hours, scanning electronmicrographs of thereconstituted human epidermis (FIG. 6) show that glycolic acid resultsin heavily damaged skin, wherein the cell envelope has been broken andthe cell cytoskeleton is clearly visible. Whilst treatment withBromelain results in a fairly smooth skin surface, there is stillevidence of rough patches. However, treatment with the hatching fluidcomposition of the invention results in an ultra smooth skin surfacethat is better than control (i.e. untreated) skin. Thus, the hatchingfluid composition may be viewed as providing a skin smoothing effect.

Light microscopy of sections of reconstructed human epidermis (FIG. 7)shows that exposure to glycolic acid for 24 hours results in thedestruction living cells, including epidermal stem cells from thestratum basale. All that remains following treatment with glycolic acidare a few pyknotic nuclei (where the chromatin has condensedirreversibly) and anucleated cells. Exposure to Bromelain for 24 hoursresults in minimal alterations to the stratum basale and the stratumspinosum. In contrast, after exposure to the hatching fluid compositionof the invention for 24 hours the reconstructed skin shows evidence ofcell proliferation and differentiation (see arrow A of FIG. 7). After 48hours of exposure to the hatching fluid composition the skin showsevidence of a denser stratum granulosum and a higher concentration oflamellar granules (see arrow B of FIG. 7). Thus the hatching fluidcomposition of the invention has a skin rejuvenating effect and resultsin an improvement of skin barrier function.

EXAMPLE 4: ANALYSIS OF NON-SALMONIDAE HATCHING FLUID

Hatching fluid from non-Salmonidae fish species was analysed todetermine whether it contains polypeptides that are equivalent to thepolypeptides found in cosmetic compositions derived from Salmonidaehatching fluid.

An antibody was raised against a principle polypeptide component of thehatching fluid compositions described in Examples 1-3. The antibody wasused to determine whether similar polypeptides are present in hatchingfluid from Medaka (Oryzias latipes) and Zebrafish (Danio rerio).

Salmon hatching fluid was obtained according to the method described inExample 1.

Hatching fluid from Medaka and Zebrafish was obtained from the ZebrafishLaboratorium at the Department of Biology at the University of Bergen.In this respect, a sample of Zebrafish hatching fluid (30 mL) wasobtained from water in which approximately 100 embryos had hatched. Asample of Medaka hatching fluid (14 mL) was obtained from water in whichapproximately 14 embryos had hatched. Water from an aquarium was used asa Blank control.

The samples were concentrated by freeze drying for 48 hours(approximately 10 fold) before analysis. The protein concentration ofthe concentrated samples was measured in a Spectrophotometer at A280 nm.The absorbance of the samples was: Salmon A280=5.6; Medaka A280=1.6; andZebrafish A280=0.1.

The concentrated polypeptide samples were analyzed by SDS-PAGE. Theresultant gels were stained by Coomassie blue or analyzed by Westernblotting, using the aforementioned antibody against a principlecomponent of the Salmon hatching fluid compositions.

The Coomassie stained gel in FIG. 8A shows several clear bands for theMedaka sample (lane 2), which are comparable to the bands observed inthe Salmon sample (lane 1). No visible bands were observed in the Blanksample (not shown) or the Zebrafish sample (lane 3). However, theabsence of visible bands in the Zebrafish sample can be explained by therelatively low concentration of protein in the Zebrafish sample.Similarly, the presence of additional bands in the Salmon sample can beexplained by the high concentration of protein in the Salmon sample,which means that even less abundant proteins are visible.

FIG. 8B shows a Western blot which demonstrates that the antibody reactsspecifically with a single band in the Salmon sample and cross-reactedwith a protein in both the Medaka sample and the Zebrafish sample.Again, the difference in the intensity of the bands can be explained bythe different concentration of protein in these samples.

The polypeptide identified in the Salmon sample was approximately 25 kDa(lane 1). The cross-reactive polypeptide in the Medaka sample wasapproximately 35 kDa (lane 2) and approximately 27 kDa in the Zebrafishsample (lane 3). No polypeptides were detected by the antibody in theBlank sample (not shown). Whilst not wishing to be bound by theory, thedifference in the size of the polypeptides from Salmon, Medaka andZebrafish may be attributed to interspecies variation and it is notuncommon for functionally equivalent polypeptides to vary in size indifferent species, for instance, due to different post-translationalmodifications such as glycosylation.

These results indicate that hatching fluid from each species contains asimilar array of polypeptides and non-Salmonidae fish species containpolypeptides that are structurally similar to the polypeptides in thecosmetic compositions described in Examples 1-3. The polypeptides innon-Salmonidae hatching fluid could be expected to have the samefunctional properties as the polypeptides in the cosmetic compositionsderived from Salmon hatching fluid. Thus, the hatching fluid fromnon-Salmonidae fish is a suitable starting material in the methodsdescribed herein and is expected to result in effective cosmeticcompositions in accordance with the invention.

The invention claimed is:
 1. A method for reducing or preventing the cosmetic appearance or prevalence of wrinkles, fine lines, hyperpigmentation, laxity, dry skin, scaling and/or transepidermal water loss in skin of a mammalian animal, comprising administering a cosmetic composition to said animal, wherein said composition is obtained or obtainable by a method consisting of the steps of: a) suspending fish eggs, wherein said eggs are not Salmonidae eggs, in a minimal volume of water; b) inducing synchronized, rapid hatching of said eggs; c) optionally filtering the hatched eggs to obtain hatching fluid; and d) filtering the hatching fluid of b) or c) to obtain the composition, wherein the step of filtering the hatching fluid consists of the steps of: (i) filtering the hatching fluid using a filter with a pore size of at least 5 μm, and collecting the filtrate; (ii) filtering the filtrate from step (i) using a filter with a pore size of 0.30-0.60 μm, and collecting the filtrate; (iii) exchanging the water in the filtrate from step (ii) with a pharmaceutically acceptable buffer by performing diafiltration using a filter with an exclusion size of less than 15 kDa; (iv) optionally repeating the diafiltration step of step (iii); (v) optionally diluting the solution from step (iii) or (iv); (vi) filtering the solution from step (iii), (iv) or (v) using a filter with a pore size of 0.15-0.30 μm, and collecting the filtrate, wherein the filtrate is substantially free of components derived from the eggs other than polypeptides or portions thereof; and (vii) preparing said cosmetic composition from the filtrate from step (vi).
 2. The method of claim 1, wherein said method is for reducing or preventing the cosmetic appearance or prevalence of wrinkles and/or laxity.
 3. The method of claim 1, wherein: a) the pore size of the filter in step (i) is 5-15 μm; b) the pore size of the filter in step (ii) is 0.35-0.55 μm; and/or c) the pore size of the filter in step (vi) is 0.22 μm.
 4. The method of claim 1, wherein the eggs are from a fish selected from a fish of any Superorder selected from the list consisting of Osteoglossomorpha, Elopomorpha, Clupeomorpha, Ostariophysi, Protacanthopterygii (excluding fish from the Salmonidae family), Stenopterygii, Cyclosquamata, Scopelomorpha, Lampridiomorpha, Polymyxiomorpha, Paracanthopterygii and Acanthopterygii.
 5. The method of claim 4, wherein the fish is from any Order selected from the list consisting of Osteoglossiformes, Hiodontiformes, Elopiformes, Albuliformes, Notacanthiformes, Anguilliformes, Saccopharyngiformes, Clupeiformes, Gonorynchiformes, Cypriniformes, Characiformes, Gymnotiformes, Siluriformes, Argentiniformes, Salmoniformes (excluding fish from the Salmonidae family), Esociformes, Osmeriformes, Ateleopodiformes, Stomiiformes, Aulopiformes, Myctophiformes, Lampriformes, Polymixiiformes, Percopsiformes, Batrachoidiformes, Lophiiformes, Gadiformes, Ophidiiformes, Mugiliformes, Atheriniformes, Beloniformes, Cetomimiformes, Cyprinodontiformes, Stephanoberyciformes, Beryciformes, Zeiformes, Gobiesociformes, Gasterosteiformes, Syngnathiformes, Synbranchiformes, Tetraodontiformes, Pleuronectiformes, Scorpaeniformes Perciformes and Acipenseriformes.
 6. The method of claim 5, wherein the fish is from any Family selected from the list consisting of Cyprinidae, Cichlidae, Pangasiidae, Sciaenidae, Serranidae, Carangidae, Sparidae, Lateolabracidae, Moronidae, Mugilidae, Latidae, Eleotridae and Acipenseridae.
 7. The method of claim 6, wherein the fish is a species selected from the list consisting of Grass carp (Ctenopharyngodon idella), Silver carp (Hypophthalmichthys molitrix), Catla (Catla catla), Common carp (Cyprinus carpio), Bighead carp (Hypophthalmichthys nobilis), Crucian carp (Carassius carassius), Nile tilapia (Oreochromis niloticus niloticus), Pangas catfish (Pangasius pangasius), Roho labeo (Labeo rohita), Large yellow croaker (Larimichthys crocea), Greasy grouper (Epinephelus tauvina), Japanese amberjack (Seriola quinqueradiata), Gilthead seabream (Sparus aurata), Japanese seabass (Lateolabrax japonicus), European seabass (Dicentrarchus labrax), Silver seabream (Chrysophrys auratus), Flathead grey mullet (Mugil cephalus), Barramundi (Lates calcarifer), Marble goby (Oxyeleotris marmorata), Mozambique tilapia (Oreochromis mossambicus), Siberian sturgeon (Acipenser baerii) and Danube sturgeon (Acipenser gueldenstaedtii).
 8. The method of claim 1, wherein said skin is aged skin.
 9. The method of claim 1, wherein the skin of said animal is moisturized.
 10. The method of claim 1, wherein said cosmetic composition is for topical administration to the skin.
 11. A method of preparing a cosmetic composition consisting of the steps of: a) suspending fish eggs, wherein said eggs are not Salmonidae eggs, in a minimal volume of water; b) inducing synchronized, rapid hatching of said eggs; c) filtering the hatched eggs to obtain hatching fluid; and d) filtering the hatching fluid of c) to obtain the composition, wherein the step of filtering the hatching fluid consists of the steps of: (i) filtering the hatching fluid using a filter with a pore size of at least 5 μm, and collecting the filtrate; (ii) filtering the filtrate from step (i) using a filter with a pore size of 0.30-0.60 μm, and collecting the filtrate; (iii) exchanging the water in the filtrate from step (ii) with a pharmaceutically acceptable buffer by performing diafiltration using a filter with an exclusion size of less than 15 kDa; (iv) optionally repeating the diafiltration step of step (iii); (v) optionally diluting the solution from step (iii) or (iv); (vi) filtering the solution from step (iii), (iv) or (v) using a filter with a pore size of 0.15-0.30 μm, and collecting the filtrate, wherein the filtrate is substantially free of components derived from the eggs other than polypeptides or portions thereof; and (vii) preparing said cosmetic composition from the filtrate from step (vi).
 12. The method as claimed in claim 1, wherein hatching is complete within less than 2 hours for more than 95% of the embryos.
 13. The method of claim 1, wherein said cosmetic composition is coated, impregnated or chemically bonded onto a product, material or device.
 14. The method as claimed in claim 11, wherein hatching is complete within less than 2 hours for more than 95% of the embryos.
 15. The method of claim 1, wherein said preparation in step (vii) consists of the addition of one or more pharmaceutically acceptable excipients, diluents, components and/or ingredients.
 16. The method of claim 11, wherein said preparation in step (vii) consists of the addition of one or more pharmaceutically acceptable excipients, diluents, components and/or ingredients.
 17. The method of claim 1 wherein the hatching in step b) is complete within less than 6 hours for more than 80% of the embryos.
 18. The method of claim 11, wherein the hatching in step b) is complete within less than 6 hours for more than 80% of the embryos.
 19. The method of claim 1, wherein the filtrate of step (vi) consists of a solution of polypeptides or portions thereof.
 20. The method of claim 11, wherein the filtrate of step (vi) consists of a solution of polypeptides or portions thereof. 